Large ion channels in cell and organelle membranes are not only the gateways of metabolite exchange between different cellular compartments and cells; they are also recognized as multifunctional membrane receptors and can be formed by components of many toxins. To study these channels under precisely controlled conditions, we purify and then reconstitute channel-forming proteins into planar lipid bilayers.[unreadable] [unreadable] I. High-affinity blocking of anthrax and other toxin channels. Many pathogens use the formation of trans-membrane pores in target cells in the process of infection. A great number of pore-forming proteins, both bacterial and viral, are considered to be important virulence factors. This makes them attractive targets for the discovery of new therapeutic agents. Recently, using structure-inspired drug design, we demonstrated that aminoalkyl derivatives of beta-cyclodextrin inhibited anthrax lethal toxin (LeTx) action by blocking the trans-membrane pore formed by the protective antigen (PA) subunit of the toxin. Now, as a next step, we evaluate a series of new beta-cyclodextrin derivatives with the goal of identifying potent inhibitors of anthrax toxins. Newly synthesized hepta-6-thioaminoalkyl and hepta-6-thioguanidinoalkyl derivatives of beta-cyclodextrin with the alkyl spacers of various lengths were tested for the ability to inhibit cytotoxicity of LeTx in cells as well as to block ion conductance through PA channels reconstituted in planar bilayer lipid membranes. Most of the tested derivatives were protective against anthrax LeTx action at low or sub-micromolar concentrations. They also blocked ion conductance through PA channels at concentrations as low as 0.1 nM. The activity of the derivatives in both cell protection and channel blocking was found to depend on the length and chemical nature of the substituent groups. One of the compounds was also shown to block the edema toxin activity. To test the broader applicability of this approach, we sought beta-cyclodextrin derivatives capable of inhibiting the activity of Staphylococcas aureus alpha-Hemolysin (HL), which is regarded as a major virulence factor playing an important role in staphylococcal infection. We identified several amino acid derivatives of beta-cyclodextrin that inhibited the activity of HL and LeTx in cell-based assays at low micromolar concentrations. One of the compounds was tested for the ability to block ion conductance through the pores formed by HL and PA in artificial lipid membranes. We anticipate that this approach can serve as the basis for a structure-directed drug discovery program to find new and effective therapies against various pathogens that utilize pore-forming proteins as virulence factors.[unreadable] [unreadable] II. Regulation of VDAC by non-lamellar lipids of mitochondrial membranes. Evidence accumulates that lipids play important roles in permeabilization of the mitochondria outer membrane (MOM) at the early stage of apoptosis. Lamellar phosphatidylcholine (PC) and non-lamellar phosphatidylethanolamine (PE) lipids are the major membrane components of the MOM. Cardiolipin (CL), the characteristic lipid from the mitochondrial inner membrane, is another non-lamellar lipid recently shown to play a role in MOM permeabilization. We investigate the effect of these three key lipids on the gating properties of voltage-dependent anion channel (VDAC), the major channel in MOM. We find that PE induces voltage asymmetry in VDAC current-voltage characteristics by promoting channel closure at cis-negative applied potentials. Significant asymmetry is also induced by CL. The observed differences in VDAC behavior in PC and PE membranes cannot be explained by differences in the insertion orientation of VDAC in these membranes. Rather, it is clear that the two non-lamellar lipids affect VDAC gating. Using gramicidin A channels as a tool to probe bilayer mechanics, we show that VDAC channels are much more sensitive to the presence of CL than it could be expected from the experiments with gramicidin channels. We suggest that this is due to the preferential insertion of VDAC into CL-rich domains. We propose that the specific lipid composition of the mitochondria outer membrane and/or of contact sites might influence MOM permeability by regulating VDAC gating.[unreadable] [unreadable] III. Physics of channel-facilitated metabolite transport. The past years progress in quantitative understanding of channel-facilitated transport resulted in the following findings. 1) To probe the size of the ion channel formed by Pseudomonas syringae lipodepsipeptide Syringomycin E, we used the partial blockage of ion current by penetrating poly(ethylene glycol)s. Earlier experiments with symmetric application of these polymers yielded a radius estimate of 1 nm. Now, motivated by the asymmetric non-ohmic current-voltage curves reported for this channel, we explored its structural asymmetry. We gauged this asymmetry by studying the channel conductance after one-sided addition of differently sized poly(ethylene glycol)s. We have found that small polymers added to the cis-side of the membrane (the side of lipodepsipeptide addition) reduce channel conductance much less than do the same polymers added to the trans-side. We interpret these results to suggest that the water-filled pore of the channel is conical with cis- and trans-radii differing by a factor of 23 and that the smaller cis-radius is in the 0.250.35 nm range. In symmetric, two-sided addition, polymers entering the pore from the larger opening dominate blockage. 2) We studied the distribution of direct translocation times for particles passing through membrane channels between two reservoirs. The direct translocation time is a conditional first-passage time defined as the residence time of the particle in the channel while passing through the membrane directly, i.e., without returning to the reservoir from which it entered. We have shown that the distributions of direct translocation times are identical for translocation in both directions, independent of any asymmetry in the potential across the channel and, hence, the translocation probabilities. 3) Channel-forming proteins in a lipid bilayer of a biological membrane usually respond to variation of external voltage by changing their conformations. Periodic voltages with frequency comparable with the inverse relaxation time of the protein produce hysteresis in the occupancies of the protein conformations. If the channel conductance changes when the protein jumps between these conformations, hysteresis in occupancies is observed as hysteresis in ion current through the channel. We have developed an analytical theory of this phenomenon assuming that the channel conformational dynamics can be described in terms of a two-state model. The theory describes transient behavior of the channel after the periodic voltage is switched on as well as the shape and area of the stationary hysteretic loop as functions of the frequency and amplitude of the applied voltage.